Shukuan Xu

Assessment of fluorescence resonance energy transfer for two-color DNA microarray platforms.

Two-color DNA microarray platforms are widely used for determining differential amounts of target sequences in parallel between sample pairs. However, the fluorescence (or Forster) resonance energy transfer (FRET) between two fluorophores can potentially result in the distortions of the measured fluorescence signals. Here we assessed the influence of FRET on the two-color DNA microarray platform and developed a reliable and convenient method for the correction of FRET distortion. Compared to current methods of normalization based on the statistical analysis and the hypothesis that only a small part of target sequences are differentially presented between sample pairs, our FRET correction method can recover the undistorted signals by the compensation of fluorescence emission, without considering the number of target sequences differentially presented. The correction method was validated with samples at different target ratios and with microarrays spotted in different probe concentrations. We also applied the FRET correction method to gene expression profiling arrays, and the results show that FRET was present when the content of target sequence was beyond a threshold amount and that the process incorporating our FRET correction method can improve the reliability of the gene expression profiling microarray platform in comparison with the current process without FRET correction.

Digital imaging scanning system and biomedical applications for biochips

Biochips have been an advanced technology for biomedical applications since the end of the 20th century. Optical detection systems have been a very important tool in biochip analysis. Microscopes are often inadequate for high resolution and big view-area detection of microarray chips, thus some new optical instruments are required. In this work, a novel digital imaging scanning system with dark-field irradiation is developed for some biomedical applications for microarray chips, characterized by analyzing genes and proteins of clinical samples with high specific, parallel, and nanoliter samples. The novel optical system has a high numerical aperture (NA=0.72), a long working distance (wd>3.0 mm), an excellent contrast and signal-to-noise ratio, a high resolving power close to 3 mum, and an efficiency of collected fluorescence more than two-fold better than that of other commercial confocal biochip scanners. An edge overlap algorithm is proposed for the image restructure of free area detection and correcting scanning position errors to a precision of 1 pixel. A novel algorithm is explored for recognizing the target from the scanning images conveniently, removing noise, and producing the signal matrix of biochip analysis. The digital imaging scanning system is equally as good for the detection of enclosed biochips as it is for the detection of biological samples on a slide surface covered with a glass cover slip or in culture solution. The clinical bacteria identification and serum antibody detection of biochips are described.

Real‐time and label‐free detection of chloramphenicol residues with specific molecular interaction

Molecular measurements with specific or non‐specific molecular interaction are usually reported by using different methods. This paper shows a method for small‐molecular label‐free measurements in real time with specific and non‐specific molecular interaction based on surface plasmon resonance. The quantitative measurement of glucose with non‐specific molecular interaction was performed simply to validate our self‐built angle‐dependent surface plasmon resonance system, which showed a good sensitivity and stability, such as that of Fourier transform infrared spectrometer. As application, we present the measurement of chloramphenicol residues with specific molecular interaction employing an indirect competitive immunoreaction by using the same surface plasmon resonance system with a limit of detection about 0.5 ng mL−1, which shows a potential application for food safety.

Quantitative fluorescence correction incorporating Förster resonance energy transfer and its use for measurement of hybridization efficiency on microarrays.

Fluorescence detection using two spectrally distinct fluorophores has long been used for the determination of the relative abundance of biomolecules, but overlap between the fluorescence spectra of each fluorophore can result in nonradiative Förster resonance energy transfer (FRET) and distorting the signals detected by fluorescence channels. Thus conventional methods for quantifying the relative abundance of fluorophores by fluorescence emission will not be accurate if FRET can occur. In this paper we report the development of a quantitative fluorescence correction method incorporating FRET to measure the relative abundance of fluorophores in dual-labeling experiments. The quantitative fluorescence correction method incorporating FRET is accurate, comprehensive, and convenient for the measurement of the relative abundance of fluorophores in dual-labeling experiments and can also correct the FRET distortion and provide accurate, quantitative, and convenient measurement of the hybridization efficiencies on microarrays.

Development of a confocal optical system design for molecular imaging applications of biochip

A novel confocal optical system design and a dual laser confocal scanner have been developed to meet the requirements of highly sensitive detection of biomolecules on microarray chips, which is characterized by a long working distance (wd>3.0 mm), high numerical aperture (NA=0.72), and only 3 materials and 7 lenses used. This confocal optical system has a high scanning resolution, an excellent contrast and signal-to-noise ratio, and an efficiency of collected fluorescence of more than 2-fold better than that of other commercial confocal biochip scanners. The scanner is as equally good for the molecular imaging detection of enclosed biochips as for the detection of biological samples on a slide surface covered with a cover-slip glass. Some applications of gene and protein imagings using the dual laser confocal scanner are described.

DETECTION AND APPLICATION OF MICROFLUIDIC ISOTHERMAL AMPLIFICATION ON CHIP

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method. Compared with the widely utilized polymerase chain reaction (PCR), LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures. In this paper, we designed and fabricated different kinds of polycarbonate (PC) microfluid chips, explored appropriate reaction condition for LAMP in microenvironment (1 nL → 10 μL), and developed a microfluidic isothermal amplification detection system. The DNA optimal amplification temperature is obtained; the starting time of exponential amplification of DNA is put forward farther. The optimal condition of DNA amplification in microenvironment, with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.

LABEL-FREE DETECTION OF PROTEIN MICROARRAY WITH HIGH THROUGHPUT SURFACE PLASMON RESONANCE IMAGING (SPRI)

A surface plasmon resonance imaging (SPRI) system was developed for the discrimination of proteins on a gold surface. As a label-free and high-throughput technique, SPRI enables simultaneously monitoring of the biomolecular interactions at low concentrations. We used SPRI as a label-free and parallel method to detect different proteins based on protein microarray. Bovine Serum Albumin (BSA), Casein and Immunoglobulin G (IgG) were immobilized onto the Au surface of a gold-coated glass chip as spots forming a 6 × 6 matrix. These proteins can be discriminated directly by changing the incident angle of light. Excellent reproducibility for label-free detection of protein molecules was achieved. This SPRI platform represents a simple and robust method for performing high-sensitivity detection of protein microarray.

Microsphere-enhanced label-free high-throughput molecular detection

In this paper, we developed a microsphere enhanced and label free high throughput molecular detection based on SPRI and microarray chips, and a self-built surface plasmon resonance imaging instrument was set. One label-free protein chip forming a 7× probe array was designed and fabricated using a commercial microarray robot spotter on chemical modified gold-coated glass slide. Antibody molecules were successfully detected in label-free and high-throughput method by using this chip. The detection signals on the chip was successfully enhanced by using microspheres with a diameter of 1 μm.

Single Living Cell Imaging and Spectrum Detection

Microscopy is an important tool in biology and medicine, but it is often limited to high numerical aperture (NA) with a short working distance(wd), such as NA = 0.6 then wd < 1 mm. For imaging living cells in culture, a high numerical aperture and long working distance of optical imaging structure is required, and the common microscopy objective is no good here. In order to meet the single living cell imaging, a novel optical design has been performed in this paper, which is character of ultra-long working distance wd = 15 mm and high numerical aperture NA = 0.7. An optical imaging system with dual modes information of fluorescence and spectrum can be developed for the subcellular imaging of cells attached on surface of vessel or free-floating in culture.

A Novel Developed Detection and Analysis Method of DNA Microarray Hybridization Using FRET technique

Fluorescence resonance energy transfer (FRET) is a distance-sensitive energy transfer process which is widely used in probing molecular interaction in the 1-10 nm range. DNA microarray chip technique is a high throughput analysis method in molecular biology research. In this paper, we explored a novel detection and analysis method of DNA microarray hybridization using FRET technique. In our study, TMR dye labeled DNA oligomer was immobilized on the microarray chips and hybridized with complementary DNA oligomer labeled with Cy5 dye. We successfully detected and analysed the FRET signal of the hybridized DNA microarray. The variation of FRET signal intensity and the efficiency of FRET in response to the concentration of the sample were studied.